Letrozole

People in the DOR-fresh group using GnRH antagonist protocol. Patients using GnRH antagonist protocol started 300–450 IU recombinant FSH (Gonal-F; Merck Serono) or follitropin β (Puregon®; Organon) either alone or in combination with human menopausal gonadotropin (Menopur; Ferring) on days 2–3 of the menstrual cycle. Subcutaneous cetrorelix (Cetrotide; Merck Serono) 0.25 mg was introduced daily as soon as follicles reached 14 mm in diameter until trigger day. The gonadotropin dosage was adjusted every 2–3 days in accordance with follicle growth. When the leading follicle reached 16–18 mm in diameter, final oocyte maturation was induced with the combination of 250 μg recombinant hCG (Ovidrel; Merck Serono) and 0.2 mg triptorelin (Decapeptyl; Ferring). Transvaginal oocyte retrieval was performed under transvaginal ultrasound guidance 35 to 36 h after triggering.
People in the DOR-Accu group used double stimulations in the same menstrual cycle with gonadotropins with or without clomiphene or letrozole combination. In follicle phase stimulation, clomiphene citrate 150 mg/day or letrozole 7.5 mg/day were given on days 2–3 of the menstrual cycle. Gonadotropin 150–450 IU/day was added later when three or more follicles reached 10 mm in diameter, and 0.25 mg subcutaneous cetrorelix (Cetrotide; Merck Serono) was administered in the presence of 14 mm follicle until trigger day. When the leading follicle reached 16–18 mm in diameter, oocyte maturation was triggered, and oocytes were retrieved as the DOR-fresh group. Transvaginal ultrasound was performed after oocyte retrieval, and clomiphene citrate 150 mg/day or letrozole (7.5 mg/day) was given in the presence of at least one AFC. Gonadotropin 150–450 IU/day was added when three or more follicles reached 10 mm until trigger day. Administration of GnRH antagonist, the trigger of oocyte maturation, and oocyte retrievals were carried out as the follicular phase.
The oocytes from the DOR-Accu group were vitrificated by a 3-step gradient cryoprotectant loading process using Cryotec Vitrification Method® (REPROLIFE Inc. 2–5-3-9F Shinjuku, Tokyo, Japan). The oocytes were equilibrated for 12–15 min in a 0.3 ml equilibration solution. Then oocytes were washed in a 0.3 ml vitrification solution (VS) for 30–40 secs and replaced with new 0.3 ml VS for another 10–20 secs. In the next step, oocytes were loaded on the Cryotec seat with minimum (0.01–0.1 μl) VS volume and immediately submerged Cryotec into liquid nitrogen directly and then covered with a cap. All procedure was performed at room temperature (25 °C–27 °C) with media being prepared at least one hour in advance.
Firstly, the warming procedure (REPROLIFE Inc. 2–5-3-9F Shinjuku, Tokyo, Japan) started with Cryotec removal from liquid nitrogen and immersion in 1 ml warming solution (TS) for 1 min. Secondly, oocytes were transferred into a 0.3 ml dilution solution for 3 min. Thirdly, oocytes were equilibrated in 0.3 ml washing solution (WS) for another 5 min and replaced with a new 0.3 ml WS for another 1 min. Finally, oocytes were incubated in culture media for two hours at 37 °C, 6.0% CO2, and 5% O2 before intracytoplasmic sperm injection (ICSI). TS was placed in an incubator at 37 °C at least three hours before use, and the other two were prepared for one hour at room temperature (25 °C–27 °C) in advance. All warming procedure was also performed at room temperature.

In this experimental study, a total of 20 female Sprague-Dawley rats (130-180 gr, 8 wk) were obtained from the Center of Comparative and Experimental Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. Female rats were maintained under standard conditions (12 hr light/dark cycle, temperature of 23 $\pm$ 3 C, and relative humidity of 25 $\pm$ 5%) with free access to rodent food and water. After adaptation to the new environment, 18 female rats in the proestrous or estrous phase were joined with sexually experienced adult male rats (mean weight: 375 gr) with a 2:1 female/male ratio overnight. In the morning after mating, a vaginal plug or spermatozoa in the vaginal smear was considered as the first day of pregnancy (GD1). 15 pregnant rats were randomly selected for letrozole administration during pregnancy.

Pregnant rats were housed in standard plastic cages individually. They were randomly divided into 4 experimental groups (n = 3/each) to orally receive letrozole at 4 doses (0.25, 0.75, 1.00, and 1.25 mg/kg BW) and a control group. Considering the reports on the embryotoxic effects of letrozole on pregnant rats and rabbits (16), a pilot study was performed to identify the doses above the physiological levels that were not lethal to the mother or the offspring. Accordingly, letrozole administration between 1.5 and 3.0 mg/kg BW caused fetal mortality, adsorption or death in early life, and uterine infection.
Letrozole (L6545, Sigma-Aldrich, St. Louis, USA) was dissolved in 1% carboxymethylcellulose (C5013, Sigma-Aldrich, St. Louis, USA) and was orally administered on days 16-18 of gestation (13). Testicular testosterone surge on 16-18 GDs is necessary for the brain masculinization and normal development of male rats (9, 10). The control rats were administrated with 1% carboxymethylcellulose on same days. The offspring's number, birth weight, and sex were recorded. The offspring remained with their mothers until weaning. At postnatal day (PND) 21, they were weaned, sexed, and weighed, and their AGD was measured. The anogenital distance index (AGDI) was calculated as AGD/BW $\times$ 100 (17). Male offspring (n = 4 per group) were kept in separate standard cages and were weighed weekly until the end of the study.